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1. BIOASSAY TECHNIQUES

2. Biological assessment.
• Estimation or determination of concentration or potency
of a physical, chemical or biological substance (agent)
by means of measuring and comparing the magnitude of
the response of the test with that of standard over a
suitable biological system under standard set of
conditions.
• The estimation of the concentration or potency of a
substance by measurement of the biological response
that it produces.
• The structure of bioassay:
STIMULUS-applied to subject.
RESPONSE-of the subject to the stimulus.

3. The purpose of bioassay
• To ascertain the potency of a drug and hence it serves
as the quantitative part of any screening procedure
(Research).
• To standardize drugs, vaccines, toxins or poisons,
disinfectants, antiseptics etc., so that each contains
the uniform specified pharmacological activity.
• Helps to determine the specificity of a compound to be
used e.g. Penicillin's are effective against Gram +ve but
not on Gram -ve
• Certain complex compounds like Vitamin B-12 which
can't be analysed by simple assay techniques can
be effectively estimated by Bioassays.
• For samples where no other methods of assays are
available.

4. Principle of Bioassay
• Bioassay involves the comparison of the main pharmacological response of
the unknown preparation with that of the standard.
• The reference standard and test sample should have same pharmacological
effect and mode of action, so that their DRC curve run parallel and their
potency ratio can be calculated.
• The test solution and standard should be compared for their established
pharmacological effect using a specified pharmacological technique.
• The method selected should be reliable, sensitive, and reproducible and
should minimize errors due to biological variation and methodology. .

5. Need of Bioassay
• They not only help to determine the concentration but also the potency of the
sample. Potency denotes activity of the compound i.e. if a compound shows
better activity at minute concentration, greater is the potency, and if its
activity is low at higher concentrations, lesser is the potency.
• It is used to standardize drugs, vaccines, toxins/ poisons, disinfectants,
antiseptics etc. as these are all used over biological system in some form.
• Sometimes the chemical composition of samples is different but has same
biological activity.
• For samples where no other methods of assays are available.

6. The standards are internationally accepted samples of drugs maintained and
recommended by the Expert Committee of the Biological Standardization of
W.H.O.
They represent the fixed units of activity (definite weight of preparation) for drugs.

7. In India
• Standard drugs are
maintained in
Government
institutions like
1. Central Drug
Research Institute,
Lucknow
2. Central Drug
Laboratory, Kolkata.

8. Classification of
bioassay.
• In-vitro
• In-vivo
• Ex-vivo

10. In vitro techniques
• These techniques employ a cell culture of
recommended biological system to study the
effect of compound under standard
condition not similar to that of living
environment. Here the cell culture survives by
utilization of the nutrition in the media.
• Ex: use of stem cells,
cell culture,
microbes (bacteria) etc.

11. In vivo techniques:
• These techniques employ a
living animal recommended
for the purpose of assay.
The techniques aims to
study the biological effect
or response of the
compound under screening
in a living system directly.
• Ex: By use of rodents,
rabbits etc.

12. Ex vivo techniques:
• These techniques employ a tissue
or cells of recommended living
system to study the effect of
compound under test in suitable
conditions within the stipulated
time of organ survival outside
the body.
• Ex: Use of any isolated organ from
animals in a glass ware to study
the effect of compound within the
period of its survival outside the
living body with provision of only
oxygen, glucose and isotonic salts
to maintain cell & cell organelles
integrity.

13. Types of bioassay
• Qualitative bioassay is used for assessing the physical
effects of a substance that may not be quantified, such
as abnormal development or deformity.
eg: Arnold Adolph Berthold's famous experiment
on castrated chickens. This analysis found that
by removing the testes of a chicken, it would not
develop into a rooster because the endocrine
signals necessary for this process were not
available.
• Quantitative bioassays involve estimation of
concentration/potency of a substance by measurement
of the biological response it produces. These bioassays

14. 1. End Point or Quantal Assay:
• Here the threshold dose producing a positive effect is
measured on each animal and the comparison between the
average results of two groups of animals (one receiving
standard and other the test) is done.
• Bioassay of digitalis in cats.
• Potency is calculated as follows:

15. Bioassay Methods.
• 2. Graded Response Assay: : It is
proportional to the dose and response may
lie between no response and maximum
response.
• This method is based on the assumption of
the dose-response relationship. Log-dose-
response curve is plotted and the dose of
standard producing the same response as
produced by the test sample is directly read
from the graph.
• E.g. Acetyl-choline producing
contraction in the rat ileum

16. Matching point or bracketing
method• Here a constant dose of the standard is bracketed by
varying dose of sample until an exact matching
between the standard dose responses and the
particular dose response of the sample is achieved.
This technique is used
• when test sample is too small
• Inaccurate & margin of error
difficult to estimate
Eg: histamine on guinea pig ileum,

17. Interpolation assay.
• Bioassays are conducted by determining the
amount of preparation of unknown potency
required to produce a definite effect on
suitable test animals/organs/Tissue under
standard conditions.
• This effect is compared with that of a
standard. Thus the amount of the test
substance required to produce the same
biological effect as a given quantity the unit
of a standard preparation is compared and
the potency of the unknown is expressed as
a % of that of the standard by employing a
simple formula.

18. Multi point Bioassay.
• This method incorporates the
principle of interpolation and
bracketing.
• 2+1 indicates- Two response of
Standard and one response of Test
respectively.
• This procedure of 2+1 or 2+2 is
repeated 3 times or 4 times based
on the method with crossing over of
all the samples.
• It can further divided as 3 point, 4
point and 6 point bioassay.

19. Three point assay [2+1 dose assay]
• Fast & convenient:
– Log dose response [LDR] curve plotted with varying conc of std
drug solutions and given test solution
– Select two std doses s1& s2 [ in 2:3 dose ratio] from linear part
of LDR [ Let the corresponding response be S1, S2]
– Choose a test dose t with a response T between S1 & S2
– Record 4 sets data as follows
• s1 s2 t
• t s1 s2
• s2 t s1
• s1 s2 t
• Log Potency ratio [M] = [(T –S1) / (S2-S1)] X log (dose ratio)

20. 4 point assay [2 +2 dose assay]
• [E.g. Ach bioassay]
• Log dose response [LDR] curve plotted with varying conc of std Ach
solutions and given test solution
• Select two std doses s1& s2 from linear part of DRC [ Let the
corresponding response be S1, S2]
• Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ;
• Also s2/s1 = t2/t1 = 2/3
Record 4 data sets
• s1 s2 t1 t2
• s2 t1 t2 s1
• t1 t2 s1 s2
• t2 s1 s2 t1

21. Other bioassay’s.
• Immunological assay (ELISA).
• Radioimmunoassay.
• Chemiluminescence

22. ELISA (immunological assay)
• ELISA is a popular format of a "wet-
lab" type analytic biochemistry assay
that uses a solid-phase enzyme
immunoassay (EIA) to detect the
presence of a substance, usually an
antigen, in a liquid sample or wet
sample.
• The ELISA has been used as a
diagnostic tool in medicine and plant
pathology, as well as a quality-
control check in various industries.
• The substances detected by ELISA
tests include hormones, bacterial
antigens and antibodies.

23. Types of ELISA.
• Direct ELISA: involve attachment of the antigen to the solid phase,
followed by an enzyme-labeled antibody. This type of assay generally
makes measurement of crude samples, since contaminating proteins
compete for plastic binding sites.
• Indirect ELISA: involve attachment of the antigen to a solid phase, but
in this case, the primary antibody is not labeled. An enzyme-
conjugated secondary antibody, directed at the first antibody, is then
added. This format is used most often to detect specific antibodies in
sera.
• Competitive ELISA: involves the simultaneous addition of 'competing'
antibodies or proteins. The decrease in signal of samples where the
second antibody or protein is added gives a highly specific result.
• Sandwich ELISA: involve attachment of a capture antibody to a solid
phase support. Samples containing known or unknown antigen are
then added in a matrix or buffer that will minimize attachment to the
solid phase. An enzyme-labeled antibody is then added for detection.

25. Applications of ELISA
• ELISA Test Applications in
• Antibody Concentration Determination
• Monoclonal Antibody Screening
• Virus test (HIV)
• Home Pregnancy Test
• Food industry (detecting potential food allergens

26. Radioimmunoassay
• RIA is a very sensitive in vitro assay technique used to measure
concentrations of antigens (E.g. hormone levels in the blood) by use
of antibodies.
• It is the estimation of the concentration of the substance in a unit
quantity of preparation using radiolabelled antigens.
• It requires special precautions and licensing, since radioactive
substances are used.
• A number of drugs are estimated now days by radioimmunoassay
methods because these methods are highly specific and highly
sensitive.
• Eg: the radioimmunoassay of insulin is based on the ability of human
insulin (unlabelled) to displace beef’s insulin (which may be labelled)
from the binding sites (i.e. antibodies).

27. Principle of radioimmunoassay
It uses an immune reaction [antigen-
antibody reaction] to estimate a ligand.
Ag+Ag*+Ab → [Ag -Ab+ Ag*Ab + Ag +
Ab*]
• - Unbound Ag* and Ag washed out
• - Radio activity of bound residue
measured.
• - Ligand concentration is inversely
related to
the radio activity.
• - [Ag: ligand to be measured;
Ag*:radiolabelled ligand].

28. Method of RIA
• Requirements:
• 1. Preparation and
characterization of an antigen
• 2. Radio labeling of the antigen
• 3. Preparation of the specific
antibody
• 4. Development of assay system.
• Components of RIA Assay Kit:
• Drug
• Antibody
• Labeled Drug

29. Applications of RIA.Endocrinology
• Insulin, HCG, Vasopressin
• Detects Endocrine Disorders
• Physiology of Endocrine Function
Pharmacology
• Morphine
• Detect Drug Abuse or Drug Poisoning
• Study Drug Kinetics
Epidemiology
• Hepatitis B
Clinical Immunology
• Antibodies for Inhalant Allergens
Oncology
• Carcino embryonic Antigen
• Early Cancer Detection and Diagnosis.

30. To Sum - up
• Bioassay & its principles,
structure.
• Types & methods of
bioassay.
• Immunological assay
(ELISA).
• Radioimmunoassay.

31. Chemiluminescence

32. LUMINESCENCE
“ Cold light” that can be emitted at lower temperature
Source kicks an electron of an atom out of its lowest energy
“ground” state into a higher energy “excited” state
 Finally electron returns the energy in the form of light so it
can fall back to its “ground” state

33. TYPES LUMINESCENCE
Excitation event process
Chemicals Luminol Isoluminol acridinium ester Chemiluminescence
Biochemical Luciferin
aequorin
Bioluminescence
Electromagnetic Ruthenium
Tris (bipyridly) chelate
Electroluminescence
Photons inorganic phosphors Photoluminescence

34. CHEMILUMINESCENCE
Emission of light with limited emission of heat (luminescence), as
the result of a chemical reaction.
[A] + [B] → [◊] → [Products] + light
[A], [B]: reactants
[◊]: excited intermediate

35. CHEMILUMINISCENCE
Luminol and peroxidase before adding
H2O2
Chemiluminiscence after addition H2O2

36. Application of Chemiluninescence
 Chemiluninescence immunoassay
 DNA detection
 Western blotting
 Forensic science
 Food analysis

37. CHEMILUMINESENCE IMMUNOASSAY
Provides a sensitive, high throughput alternative to conventional
colorimetric methodologies
Principle: -same as ELISA
-uses chemiluminescent substrate, hydrogen peroxide,
enhancers
-stopping reagent is not required
-Incubation period is small

38. • USES
Hormones: insulin, thyroxin, estradiol, testosterone,
progesterone
Vitamin: vit B12
Tumor markers: bone morphogenic protein-2, carcino
embryonic antigen (CEA), alpha fetoprotein (AFP)
Human beta chorionic gonadotropin
C-reactive protein
Tumor necrosis factor

39. DNA hybridization detection
Southern blotting Involves DNA separation, transfer and
hybridization
Hybridization - Process of forming a double-stranded DNA
molecule between a single-stranded DNA probe and a single-
stranded target DNA

40. • USES
Identifying DNA in crime case
Paternal Dispute
Classify DNAs of various organism

41. Western blotting
Western blotting (or protein immunoblotting) is a technique widely
used to detect specific proteins in samples of tissue homogenate, cell
lysates, cell culture supernatants or body fluids.

42. Western blotting

43. Forensic science
Chemiluminescence is used by criminalists to detect traces of
blood at crime scene
Solution: luminol powder (C8H7O3N3), hydrogen peroxide, and a
hydroxide (eg. KOH) sprayed where blood might found
Tiny amount of iron from Hb in blood serves as catalyst for the
chemiluminescence reaction, luminol to glow

44. A trail of blood made visible with the use of the reagent luminol

45. Food analysis
Organophosphorous most popular pesticide

46. Limitations
• Light leaks from assay reagent & reaction vessels
• Ultra sensitive – stringent controls on purity of reagents
• High intensity light emission leads to pulse pileup in
photomultiplier tubes leads to underestimation

47. THANK YOU
bob squid